1. Studies on phosphoribosylpyrophosphate (PRPP) synthetase (1) Rat PRPP synthetase subunits I and II (PRS I and PRS II, respectively) were expressed in Escherichia coli. The recombinant isoforms were purified to apparent homogeneity and characterized. The two isoforms differed in sensitivity to inhibition by ADP and GDP, and heat inactivation. (2) cDNA clones for human PRS I were isolated from a glioblastoma cell line MGC 1 cDNA library.The deduced amino acid sequences were identical between human and rat PRS I. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago. (3) The 5' regions of the human PRS I and PRS II genes were isolated and sequenced. Chloramphenicol acetyltransferase/promoter fusion assays suggest that the 5' flanking regions cloned possess the promoter activities and contribute to the cell-differential expression of these two genes. 2. Studies on changes in cytosolic free Mg^<2+> concentrations ([Mg^<2+>]_i) after mitogenic stimulation in single mouse Swiss 3T3 fibroblasts (1) Mitogen-induced changes in [Mg^<2+>]_i were measured at the single-cell level, using digital ratio imaging microscopy of the fluorescent probe mag-fura-2. (2) Stimulation of the cells with bombesin or epidermal growth factor in combination with insulin led to a significant increase in [Mg^<2+>]_i after 30-60 min. These results provide direct evidence for the mobilization of Mg^<2+> as an early cellular response to growth factors. It is possible that free Mg^<2+>-sensitive enzymes play regulatory roles in signal transduction through changes in [Mg^<2+>]_i.